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1.
Front Bioeng Biotechnol ; 11: 1192050, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-37304136

RESUMO

Non-enveloped virus-like particles (VLPs) are versatile protein nanoparticles with great potential for biopharmaceutical applications. However, conventional protein downstream processing (DSP) and platform processes are often not easily applicable due to the large size of VLPs and virus particles (VPs) in general. The application of size-selective separation techniques offers to exploit the size difference between VPs and common host-cell impurities. Moreover, size-selective separation techniques offer the potential for wide applicability across different VPs. In this work, basic principles and applications of size-selective separation techniques are reviewed to highlight their potential in DSP of VPs. Finally, specific DSP steps for non-enveloped VLPs and their subunits are reviewed as well as the potential applications and benefits of size-selective separation techniques are shown.

2.
Biotechnol Bioeng ; 119(6): 1522-1538, 2022 06.
Artigo em Inglês | MEDLINE | ID: mdl-35170757

RESUMO

Virus-like particles (VLPs) are an emerging biopharmaceutical modality with great potential as a platform technology. VLPs can be applied as gene therapy vectors and prophylactic or therapeutic vaccines. For non-enveloped VLPs, recombinant production of the protein subunits leads to intracellular self-assembly. The subsequent purification process includes VLP dis- and reassembly which aim at removing encapsulated impurities and improving particle properties. Filtration-based separation and processing has proven successful for VLPs but requires large product quantities and laborious experiments in early development stages. Both challenges can be tackled by implementation of process analytical technology (PAT) to efficiently obtain extensive process information. In this study, an existing PAT setup was extended to comprehensively monitor the diafiltration-based disassembly of hepatitis B core antigen (HBcAg) VLPs. Process-related signals were monitored in-line, while product-related signals, such as ultraviolet light (UV) spectra as well as static and dynamic light scattering (SLS and DLS), were monitored on-line. The applicability of the sensors for disassembly monitoring was evaluated under varying processing conditions. HBcAg VLP subunit concentrations were accurately predicted based on UV data using ordinary and partial least squares regression models (Q2 from 0.909 to 0.976). DLS data were used for aggregation monitoring while the SLS intensity qualitatively reflected the disassembly progress.


Assuntos
Antígenos do Núcleo do Vírus da Hepatite B , Vacinas de Partículas Semelhantes a Vírus , Difusão Dinâmica da Luz , Filtração , Vacinas de Partículas Semelhantes a Vírus/genética
3.
Biotechnol Bioeng ; 118(10): 3926-3940, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34170511

RESUMO

Virus-like particles (VLPs) are particulate structures, which are applied as vaccines or delivery vehicles. VLPs assemble from subunits, named capsomeres, composed of recombinantly expressed viral structural proteins. During downstream processing, in vivo-assembled VLPs are typically dis- and reassembled to remove encapsulated impurities and to improve particle morphology. Disassembly is achieved in a high-pH solution and by the addition of a denaturant or reducing agent. The optimal disassembly conditions depend on the VLP amino acid sequence and structure, thus requiring material-consuming disassembly experiments. To this end, we developed a low-volume and high-resolution disassembly screening that provides time-resolved insight into the VLP disassembly progress. In this study, two variants of C-terminally truncated hepatitis B core antigen were investigated showing different disassembly behaviors. For both VLPs, the best capsomere yield was achieved at moderately high urea concentration and pH. Nonetheless, their disassembly behaviors differed particularly with respect to disassembly rate and aggregation. Based on the high-throughput screening results, a diafiltration-based disassembly process step was developed. Compared with mixing-based disassembly, it resulted in higher yields of up to 0.84 and allowed for integrated purification. This process step was embedded in a filtration-based process sequence of disassembly, capsomere separation, and reassembly, considerably reducing high-molecular-weight species.


Assuntos
Proteínas do Capsídeo/química , Vacinas contra Hepatite B/química , Vacinas de Partículas Semelhantes a Vírus/química , Vírion/química , Proteínas do Capsídeo/genética , Vacinas contra Hepatite B/genética , Ultrafiltração , Vacinas de Partículas Semelhantes a Vírus/genética , Vírion/genética
4.
Artigo em Inglês | MEDLINE | ID: mdl-32671023

RESUMO

Virus-like particles (VLPs) are emerging nanoscale protein assemblies applied as prophylactic vaccines and in development as therapeutic vaccines or cargo delivery systems. Downstream processing (DSP) of VLPs comes both with challenges and opportunities, depending on the complexity and size of the structures. Filtration, precipitation/re-dissolution and size-exclusion chromatography (SEC) are potent technologies exploiting the size difference between product and impurities. In this study, we therefore investigated the integration of these technologies within a single unit operation, resulting in three different processes, one of which integrates all three technologies. VLPs, contained in clarified lysate from Escherichia coli, were precipitated by ammonium sulfate, washed, and re-dissolved in a commercial cross-flow filtration (CFF) unit. Processes were analyzed for yield, purity, as well as productivity and were found to be largely superior to a reference centrifugation process. Productivity was increased 2.6-fold by transfer of the wash and re-dissolution process to the CFF unit. Installation of a multimodal SEC column in the permeate line increased purity to 96% while maintaining a high productivity and high yield of 86%. In addition to these advantages, CFF-based capture and purification allows for scalable and disposable DSP. In summary, the developed set-up resulted in high yields and purities, bearing the potential to be applied as an integrated process step for capture and purification of in vivo-assembled VLPs and other protein nanoparticles.

5.
Biotechnol Bioeng ; 116(6): 1366-1379, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-30684365

RESUMO

Virus-like particles (VLPs) have shown great potential as biopharmaceuticals in the market and in clinics. Nonenveloped, in vivo assembled VLPs are typically disassembled and reassembled in vitro to improve particle stability, homogeneity, and immunogenicity. At the industrial scale, cross-flow filtration (CFF) is the method of choice for performing reassembly by diafiltration. Here, we developed an experimental CFF setup with an on-line measurement loop for the implementation of process analytical technology (PAT). The measurement loop included an ultraviolet and visible (UV/Vis) spectrometer as well as a light scattering photometer. These sensors allowed for monitoring protein concentration, protein tertiary structure, and protein quaternary structure. The experimental setup was tested with three Hepatitis B core Antigen (HBcAg) variants. With each variant, three reassembly processes were performed at different transmembrane pressures (TMPs). While light scattering provided information on the assembly progress, UV/Vis allowed for monitoring the protein concentration and the rate of VLP assembly based on the microenvironment of Tyrosine-132. VLP formation was verified by off-line dynamic light scattering (DLS) and transmission electron microscopy (TEM). Furthermore, the experimental results provided evidence of aggregate-related assembly inhibition and showed that off-line size-exclusion chromatography does not provide a complete picture of the particle content. Finally, a Partial-Least Squares (PLS) model was calibrated to predict VLP concentrations in the process solution. Q2 values of 0.947-0.984 were reached for the three HBcAg variants. In summary, the proposed experimental setup provides a powerful platform for developing and monitoring VLP reassembly steps by CFF.


Assuntos
Difusão Dinâmica da Luz/métodos , Filtração/métodos , Espectrofotometria Ultravioleta/métodos , Vírion , Biotecnologia/instrumentação , Biotecnologia/métodos , Cromatografia em Gel , Desenho de Equipamento , Microscopia Eletrônica de Transmissão , Estrutura Quaternária de Proteína , Vírion/química , Vírion/isolamento & purificação , Vírion/metabolismo
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